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. 2021 Sep 14;20(10):e13471. doi: 10.1111/acel.13471

FIGURE 6.

FIGURE 6

Rapamycin treatment ameliorates REST‐dependent senescence phenotype (a,b) Representative western blotting (a) and quantification (b) of phosphorylated and total S6, FOXO1, p21 and LC3I/LC3II in ΔCre‐REST and Cre‐REST transduced neurons treated with either vehicle (‐) or 30 nM rapamycin (+) for 72 h. Calnexin was used as loading control. n = 4–6 independent preparations. (c,d) Representative western blotting (c) and quantification (d) of ubiquitin and p62 levels. Calnexin was used as loading control. n = 4–6 independent preparations. (e) The mRNA levels of CtsB, CtsD, CtsS, Laptm5, and HexB were quantified by qRT‐PCR in Cre‐transduced neurons incubated either with vehicle (Ctr), rapamycin (Rap), rapamycin plus 3‐MA (Rap +3‐MA) or trehalose for 72 h. Gapdh, Actin, and GusB were used as control housekeeping genes. n = 5 independent preparations. The dotted line indicates the control condition (vehicle‐treated ΔCre‐REST neurons). (f) Bright light representative images of ΔCre‐ and Cre‐transduced neurons treated with either vehicle (‐) or 30 nM rapamycin (+) for 72 h and stained with SA‐β‐gal to label senescent cells. Scale bar, 5 µm. (g) Quantification of the number of SA‐β‐gal‐stained ΔCre‐ (blue bars) and Cre‐ (red bars) transduced neurons treated with either vehicle (‐) or 30 nM rapamycin (+) for 72 h. n = 20–25 coverslips (5 fields/coverslip) from 3 independent preparations. (h,i) Cellular ROS levels quantified by Cell Rox labeling (h) and mitochondrial ROS quantified by MitoSOX labeling (i) in ΔCre‐ and Cre‐transduced neurons treated with either vehicle (‐) or 30 nM rapamycin (+) for 72 h. n = 9 replicates from 3 independent preparations. (j) Cell death evaluated under the same experimental conditions by incorporation of propidium iodide. n = 14 coverslips (5 fields/coverslip) from 3 independent preparations. (k) The mRNA levels of Ccl3, Cxcl1, IL‐1β, IL‐6, MMP‐24 were quantified by qRT‐PCR in Cre‐transduced neurons incubated either with vehicle (Ctr), rapamycin (Rap), rapamycin plus 3‐MA (Rap +3‐MA) or trehalose for 72 h. The dotted line indicates the control condition (vehicle‐treated ΔCre‐REST neurons). Gapdh, Actin, and GusB were used as control housekeeping genes. n = 5 independent preparations. Graphs show means ± sem. *p <0.05; **p < 0.01; ***p < 0.001; ns: not significant. Two‐way ANOVA/Bonferroni's tests (b,d,g,h,i, j), one‐way ANOVA/Dunnett's tests versus vehicle (e,k)